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Hindawi International Journal of Microbiology Volume 2020, Article ID 8831322, 10 pages https://doi.org/10.1155/2020/8831322

Research Article Novel Combinations of Diverse Subclasses for Effective Suppression of Extensively Drug-Resistant -Resistant (MRSA)

Shumyila Nasir ,1 Muhammad Sufyan Vohra ,1 Danish Gul ,1 Umm E Swaiba ,1 Maira Aleem ,1 Khalid Mehmood ,2 and Saadia Andleeb 1

1Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan 2Department of Pharmacy, Abbottabad University of Science and Technology, Havelian, Pakistan

Correspondence should be addressed to Saadia Andleeb; [email protected]

Received 16 June 2020; Revised 5 October 2020; Accepted 15 October 2020; Published 29 October 2020

Academic Editor: Faham Khamesipour

Copyright © 2020 Shumyila Nasir et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. )e emergence of multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), the chief etiological agent for a range of refractory infections, has rendered all β-lactams ineffective against it. )e treatment process is further complicated with the development of resistance to glycopeptides, primary for treatment of MRSA. Antibiotic combination therapy with existing agents may provide an immediate treatment option. Minimum inhibitory concentrations (MICs) of 18 different commercially available antibiotics were determined along with their 90 possible pairwise combinations and 64 triple combinations to filter out 5 best combinations. Time-Kill kinetics of these combinations were then analyzed to find collateral bactericidal combinations which were then tested on other randomly selected MRSA isolates. Among the top 5 combinations including levofloxacin-; /clavulanic acid-; amoxicillin/clavulanic acid- cephradine; amoxicillin/clavulanic acid-ofloxacin; and /tazobactam-tobramycin, three combinations were found to be collaterally effective. Levofloxacin-ceftazidime acted synergistically in 80% of the tested clinical MRSA isolates. First-line β-lactams of lower generations can be used effectively against MRSA infection when used in combination. Antibiotics other than glycopeptides may still work in combination.

1. Introduction rendered all β-lactams ineffective against its infections. Glycopeptides (mainly ) remain the major class Hospital-acquired methicillin-resistant Staphylococcus au- of antibiotics for treatment of MRSA, but indiscriminate use reus (MRSA) has been a predominant agent for skin and of these antimicrobial agents has led to the emergence of nosocomial infections for several years [1–3]. MRSA is re- vancomycin-resistant S. aureus (VRSA) [10]. Development sponsible for about more than 20% of all bloodstream in- of resistance and other complications such as infection fections, and mortality rate is as high as 25–50% [4, 5]. recurrence, and treatment failure pose a serious hindrance in Methicillin resistance in Staphylococcus aureus is mediated treatment of MRSA infections [4, 11]. )erefore, potential by SCCmec gene, which encodes polypeptide - options for treatment of these refractory infections need to binding-protein 2a (PBP2a) [6, 7] and also provides in- be explored. Since other therapeutic interventions such as sertion sites for plasmids and transposons which assist in phage therapies are still in development, antibiotic combi- transmission of resistance to non-β-lactam antibiotics [8, 9]. nation therapy with existing antimicrobial agents provides Emergence of β-lactamase-producing MRSA strains has an immediate treatment option. 2 International Journal of Microbiology

Usage of multidrug combinations has proven successful duplicate. Synergistic combinations were determined by the against infection caused by H. pylori, M. , fractional inhibitory concentration index (FICI) method A. baumannii, P. aeruginosa, and K. [12–16]. [23–26]. FICI calculation involves MIC of the antimicrobial Historically, vancomycin- combination was agents in combination divided by the MIC of the agents successfully employed for endocarditis caused by MRSA, but alone to determine antibiotic interactions, as shown in the due to increased possibility of renal impairment, this following formula: combination is not recommended anymore [17–19]. More recent studies, where vancomycin was used concurrently MIC(comb AB) MIC(comb6 B) with beta-lactams improved in vitro results, were observed. FICI � + , (1) MIC (A) MIC(B) In another study, antibiotics from different subclasses and generations of β-lactams (, piperacillin, and whereby FICI ≤0.5 was interpreted as “synergistic,” tazobactam) were successfully combined against MRSA FICI � 1–4.0 was considered “indifferent,” and FICI >4.0 was infections [20]. inferred “antagonistic.” In this study, we combined FDA approved antibiotics For further analyses, five highly synergistic combinations from different generations and classes to determine syner- against MRSA isolate (LR-2) were selected. )e criteria for gistic combinations which do not necessarily include van- selection required antibiotics in combination to belong to comycin (or any other glycopeptide) against highly resistant lower generation with simple mechanism of action, and low MRSA. We anticipate that using antibiotic combinations of FICI values. To further confirm the synergistic mechanism of commercially available antibiotics from diverse subclasses action, Time-Kill assays were performed on selected com- has a potential for overcoming antibiotic-resistant infections binations according to a formerly described protocol [27] and may serve as a powerful technique in reversing anti- (Figure 3). Briefly, MHB was inoculated with test organism biotic resistance on top of producing bactericidal effects. and incubated to midlog phase. Aliquots containing anti- biotic combination (two or three drug combination) and test 2. Materials and Methods MRSA at density of 106 CFUs were incubated at 37°C. At times 0 h, 2 h, 4 h, 6 h, and 8 h, the inoculum was withdrawn, Isolates of S. aureus were acquired from major hospitals of serially diluted from 103 to 107, and 100 μl of each dilution Rawalpindi, Lahore, and Peshawar, and then screened for was plated on a nutrient agar medium and incubated at methicillin and vancomycin resistance. Further suscepti- optimal growth condition of S. aureus to determine colony bility testing by the Kirby disk diffusion method was per- counts [27]. Killing kinetics were interpreted as “synergistic” formed, and by using clinical breakpoints from the Clinical when ≥2-log10 reduction in growth was observed in the and Laboratory Standards Institute (CLSI), an extensively colony forming units (CFU) at 24 h with antibiotic com- drug-resistant (XDR) MRSA isolate (LR-2) was selected for bination compared to most active drug alone, and “indif- antibiotic synergy testing [21]. A total of 18 different ferent” when <2-log10 decrease was observed. commercially available antibiotics (Table 1) were used for 90 Synergistic drug combinations were then tested on other possible pairwise combinations (Figure 1(a)) and 64 triple randomly selected methicillin-resistant S. aureus isolates, combinations (antibiotic from varying class/penicillin/in- and their MIC and FICI values were determined to validate hibitor) (Figure 2(a)). Each experiment was conducted in drug combination synergy. duplicate, and mean values were calculated for each All assays were conducted in duplicate, and mean MIC combination. values were calculated along with standard deviations. )e XDR MRSA isolate was cultured in cation-adjusted FICI values were calculated from the mean MIC values Muller-Hinton Broth (MHB) for 24 hours at 37°C. Mini- obtained in individual and combination assays. )e standard mum inhibitory concentration (MIC) was determined by the deviation is represented in graphs on the error bars. broth microdilution method as described by Wiegand et al. [22]. Stock solutions of antimicrobial agents were prepared by calculating the amount of antibiotic to be used: (amount 3. Results of drug in each tablet (y))/10 mg � (amount of active agent in each tablet)/(amount of drug needed to produce 10 mg/ml Results obtained from the Kirby–Bauer disk diffusion solution (x)). method on MRSA isolate (LR-2) showed complete resistance )e antibiotic-containing media were serially diluted in to penicillin, , fluoroquinolones, amino- 96-well plate to create an array of 10-fold dilution across the glycosides, and glycopeptides and partial resistance to car- row. Log-phase culture of MRSA was diluted in MHB to bapenem, , and certain miscellaneous agents. produce 1/50 dilution of bacterial suspension and inoculated )erefore, MRSA isolate (LR-2) was classified as XDR [28]. in the 96-well plate. Column 11 and 12 were kept as negative Individual MIC assays showed insensitivity to all antibiotics and positive control, respectively. Plates were incubated for from Table 1 except for (MIC � 4 μg/ml). Double 24 hours at 37°C. Wells showing no visible turbidity after and triple antibiotic combinations from diverse subclasses 24 h were subcultured on plate count agar for colony were tested against MRSA LR-2 to identify the combinations counting. Minimum inhibitory concentration (MIC) was that were synergistic when administered concomitantly. considered as the lowest concentration inhibiting 99% of Figure 4 shows the interactions between antibiotics indi- bacterial growth. Each experiment was performed in cating synergistic or indifferent relationship. International Journal of Microbiology 3

Table 1: List of antibiotics tested against the MRSA isolate (LR-2). Antibiotic Brand name Manufacturer Form Potency Final conc. Cephalosprin Cephradine (RAD) Velosef GlaxoSmithKline (GSK) IV 1 g 10 mg/ml (CXM) Zinacef GlaxoSmithKline (GSK) IV 1.5 g 10 mg/ml Ceftazidime (CAZ) Fortum GlaxoSmithKline (GSK) IV 500 mg 10 mg/ml (CTX) Claforan Sanofi-Aventis Pak Ltd. IV 1 g 10 mg/ml (CRO) Rocephin F.Hoffmann-La Roche Ltd. Tablets 1 g 10 mg/ml (FEP) Maxipime GlaxoSmithKline (GSK) IV 1 g 10 mg/ml (AK) Gracil Sami Pharmaceticals (Pvt.) Ltd. IV 500 mg/2 ml 10 mg/ml Gentamycin (CN) Genticyn Ray Pharma (Pvt.) Ltd. IV 80 mg/2 ml 10 mg/ml Tobramycin (TOB) Nebcin AGP Limited IV 80 mg/2 ml 10 mg/ml (FOS) Monural Zambon Sachet 3 g 10 mg/ml Fluoroquinolones Ofloxacin (OFX) Oflobid Hilton Pharma (Pvt.) Ltd. Tablets 200 mg 10 mg/ml Levofloxacin (LVX) Leflox Getz Pharma Tablets 500 mg 10 mg/ml Sparfloxacin (SPX) Sparaxin Abbot Laboratories (Pak) Ltd. Tablets 100 mg 10 mg/ml Moxifloxacin (MOX) Avelox Bayer Pharma AG 10 mg/ml Meropenem (MEM) Meronem Pfizer Limited IV 500nmg 10 mg/ml Imipenem (IPM) Tienam Merck Sharp & Dohme Corp. (MSD) IV 500 mg 10 mg/ml + β-lactam Inhibitor Amoxicillin/clavulanic acid (AUG) Augmentin GlaxoSmithKline (GSK) Tablets 625 mg 10 mg/ml Piperacillin/tazobactam (TZP) Tanzo Bosch Pharmaceuticals (Pvt.) Ltd IV 4.5 g 10 mg/ml

All pairwise combinations of cephalosporin with ami- namely, tobramycin (FICI � 0.281) and fosfomycin noglycosides or carbapenem did not exhibit a synergistic (FICI � 0.281) (Figure 2(f)), piperacillin/tazobactam, and interaction. However, when combined with fluo- meropenem combination also showed synergy roquinolones, some synergy was observed especially with (FICI � 0.375). moxifloxacin (MOX/RAD FICI � 0.140; MOX/CXM )e combinations selected for further testing included FICI � 0.265; MOX/CAZ FICI � 0.140; MOX/CTX levofloxacin-ceftazidime; amoxicillin/clavulanic acid- FICI � 0.265) (Figure 1(b)). It must be noted that moxi- tobramycin; amoxicillin/clavulanic acid-cephradine; amox- floxacin is a higher generation antibiotic than levofloxacin icillin/clavulanic acid-ofloxacin; and piperacillin/tazo- [29]. An interesting pairwise synergistic combination of bactam-tobramycin. Levofloxacin combination with cephalosporin and fluoroquinolone with low FICI (0.281) cephalosporin was selected instead of moxifloxacin com- was observed between CAZ/LVX (both lower generation bination because it is a lower-generation gluoroquinolone. antibiotics) (Figure 1(c)). All double antibiotic combinations Amoxicillin/clavulanic acid and cephradine was selected of carbapenem were unsuccessful except for when combined because both antibiotics other than the inhibitors are of with fluoroquinolones. Meropenem showed synergy with lower generation. Amoxicillin/clavulanic acid showed FICI higher generation of fluoroquinolones (sparfloxacin and values lower than 0.5 with fluoroquinolones and amino- moxifloxacin with FICI of 0.132 and 0.256, respectively) glycosides, so one combination from each group was se- (Figure 1(d)), and fluoroquinolones-imipenem combination lected. Ofloxacin in combination to amoxicillin/clavulanic showed a completely synergistic interaction (FICI � 0.126) acid made this list because it is the lowest-generation (Figure 1(e)). All pairwise combinations with aminoglyco- aminoglycoside. However, tobramycin though is not a low sides were indifferent. generation aminoglycoside but showed good potential with Triple combination of amoxicillin/clavulanic acid with other penicillin-β-lactam inhibitor combinations. Hence, cephalosporin yielded a peculiar result when synergy was tobramycin in amalgamation with amoxicillin/clavulanic observed with cephradine (1st generation cephalosporin) acid and with piperacillin-tazobactam was selected. with an FICI value of 0.281 (Figure 2(b)). )is finding was Time-Kill kinetics illustrate up to 8-fold reduction in interesting because if proven successful in further experi- growth compared to most active drug alone which means a mentation, an ineffective lower generation antibiotic [30] synergistic relation exists between levofloxacin-ceftazidime; can be brought in use again. Sensitivity was observed when amoxicillin/clavulanic acid-tobramycin; and piperacillin- amoxicillin/clavulanic acid was combined with fluo- tazobactam-tobramycin (Figures 3(a)–3(e)). Bactericidal roquinolones (FICI 0.25) (Figure 2(c)) and aminoglycosides effect was observed within 8 hours of incubation in all three (FICI � 0.125) (Figure 2(d)). Triple combinations of piper- combinations. acillin/tazobactam with moxifloxacin showed synergy )e combination showing >2-log10 reduction when (FICI � 0.281) (Figure 2(e)), and with aminoglycosides, tested on other randomly selected MRSA isolates, 4 International Journal of Microbiology

MEM IPM

CAZ TOB

CRO CN

CTX FOS

CXM AK

FEP TZP

RAD AUG

LVX SPX

MOX OFX

Aminoglycoside Penicilline/β-lactam Carbapenem Synergistic Cephalosporin Indiference Fluoroquinolone Figure 1: Pairwise antibiotic testing against MRSA. (a) List of pairwise combinations. (b) MIC by broth microdilution indicates that synergistic relationship exists between moxifloxacin and lower-generation cephalosporin (FICI 0.14–0.265). (c) Levofloxacin-ceftazidime shows FICI value as low as 0.281 indicating synergy. (d) Meropenem demonstrates synergy with higher generation of fluoroquinolones, i.e., moxifloxacin (FICI 0.132) and sparfloxacin (FICI 0.265). (e) Imipenem shows collateral sensitivity with all fluoroquinolones (FICI 0.126 each). levofloxacin-ceftazidime combination, was the most suc- pressure for development of resistance. With the depleting cessful combination because collateral sensitivity was ob- arsenal of antibiotics and a gap in the successful develop- served in 80% of MRSA isolates. Both amoxicillin/clavulanic ment of alternative therapeutics, determining synergistic acid-tobramycin; piperacillin-tazobactam-tobramycin combination of currently available antibiotics could provide combination showed synergy in 50% MRSA isolates. an effective alternative to curtailing the alarming increase in antibiotic resistance [31]. 4. Discussion )e purpose of this study was to find combinations from diverse subclasses of antibiotics that can produce synergistic Rapid emergence of extensive drug resistance in Gram- effects in combinations against extremely drug-resistant positive and Gram-negative necessitates identifi- MRSA isolates. We report that a novel combination of cation of novel therapeutic approaches. Reliance on mon- levofloxacin-ceftazidime (LVX/CAZ) acts synergistically to oantibiotic therapeutic approach provided selection produce bactericidal effect on XDR MRSA isolate LR-2. International Journal of Microbiology 5

Amoxicillin/clavulanic 600 0.281 acid 500 Cephalosporin Cephradine 400 Cefuroxime Cefazidime 300 Cefotaxime Cefriaxone 200

Cefepime μ g/ml ± SD MIC 100 Aminoglycoside Amikacin 0 Gentamycin RAD CXM CAZ CTX CRO FEP Tobramycin Fosfomycin AUG Cephalosporin Fluoroquinolone AUG + cephalosporin Ofoxacin Levofoxacin Sparfoxacin Moxifoxacin Carbapenem Imipenem Meropenem Piperacillin/tazobactam Cephalosporin Cephradine Cefuroxime Cefazidime Cefotaxime Cefriaxone Cefepime

Aminoglycoside Amikacin Gentamycin Tobramycin Fosfomycin Fluoroquinolone Ofoxacin Levofoxacin Sparfoxacin Moxifoxacin Carbapenem Imipenem Meropenem

(a) (b) 600 0.25 0.25 0.25 0.25 600 0.125 0.125 0.125 0.125 500 500

400 400

300 300

200 200 MIC μ g/ml ± SD MIC MIC μ g/ml ± SD MIC 100 100

0 0 OFX LVX SPX MOX AK CN TOB FOS AUG AUG Fluoroquinolones Aminoglycosides AUG + fluoroquinolones AUG + aminoglycosides (c) (d) Figure 2: Continued. 6 International Journal of Microbiology

600 0.281 600 0.281 0.281 500 500

400 400 ± SD 300 300

200 200 MIC μ g/ml MIC MIC μ g/ml ± SD MIC 100 100

0 0 OFX LVX SPX MOX AK CN TOB FOS TZP TZP Fluoroquinolones Aminoglycosides TZP + fluoroquinolones TZP + aminoglycosides (e) (f) 80 0.375 70 60

± SD 50 10 30

MIC μ g/ml MIC 20 10 0 MEM IPM TZP Carbapenem TZP + carbapenem (g)

Figure 2: Triple antibiotic testing against MRSA. (a) List of triple combinations. (b) Amoxicillin-clavulanic acid-cephalosporin MIC indicates that synergistic relationship exists only between amoxicillin-clavulanic acid-cephradine (FICI 0.281). (c) MIC of amoxicillin- clavulanic acid-fluoroquinolone combination shows that synergy exists in all combinations (FICI 0.25 each). (d) A significant decrease in MIC of amoxicillin-clavulanic acid-aminoglycoside in combinations (FICI 0.125). (e) Reduction in combined MIC is only observed with moxifloxacin in piperacillin-tazobactam-fluoroquinolone combinations (FICI 0.281). (f) Significant activity is observed when piperacillin- tazobactam is combined with higher-generation aminoglycoside, i.e., tobramycin (FICI 0.281) and fosfomycin (FICI 0.281). (g) Meropenem in combination with piperacillin-tazobactam exhibits synergy (FICI 0.375).

Previously, fluoroquinolone-cephalosporin combination conjunction with cephradine, an ineffective lower-genera- was shown to have synergistic effect against P. aeruginosa tion antibiotic against MRSA [30], was an interesting [32]. combination. )is lower-generation antibiotic proved suc- Our findings on pairwise combinations suggest that cessful in Time-Kill studies and shows the potential to be fluoroquinolones (especially levofloxacin and moxifloxacin) used again as a therapeutic agent in combination. Although, are very effective when combined with cephalosporin and amoxicillin/clavulanic acid-tobramycin trio showed >2- carbapenem antibiotics. )ough monotherapy with fluo- log10 decrease in XDR MRSA isolate (LR-2), it did not show roquinolones is recommended to be minimized for treat- synergistic interactions in other isolates. Amoxicillin/clav- ment of S. aureus infections because of multidrug resistance ulanic acid/fluoroquinolone (ciprofloxacin) combination [33], this finding provides a solution to overcome fluo- has also been proven successful before for treatment of roquinolone resistance in MRSA. Gram-negative infections of prosthetic joints [38]. However, )e overall higher sensitivity ratios to amoxicillin/ it must be noted that most of these isolates showed low clavulanic acid in combination with other antibiotics is not individual MIC values for tobramycin and amoxicillin/ surprising since existing evidence on antimicrobial combi- clavulanic acid, and so the FICI values were mostly “in- nations suggests that despite being the first-line antibiotic, different.” Synergy between piperacillin-tazobactam-mer- beta-lactam antibiotics in combination therapy are crucial openem combination is consistent with the findings of PR for the treatment of obstinate S. aureus bacteremia [34]. Gonzales and his colleagues [20]. Besides, amoxicillin/clavulanic acid is recommended in )e inability to confirm all synergistic combinations by amalgamations to overcome vancomycin or Time-Kill assay was a limitation of our study. Due to this, we resistance in MRSA [35–37]. Amoxicillin/clavulanic acid in might have overlooked certain combinations that might International Journal of Microbiology 7

Fluoroquinolone + cephalosporin 600 0.14 – 0.265 600 Ofloxacin Cephradine 0.281 Levofloxacin Cefuroxime 500 500 Sparfloxacin Ceazidime Moxifloxacin Cefotaxime Ceriaxone 400 400 Cefepime Fluoroquinolone + aminoglycoside 300 300 Ofloxacin Amikacin Levofloxacin Gentamycin 200 200

Sparfloxacin Tobramycin μ g/ml ± SD MIC μ g/ml ± SD MIC Moxifloxacin Fosfomycin 100 100 Cephalosporin + aminoglycoside Cephradine Amikacin 0 0 Cefuroxime Gentamycin RAD CXM CAZ CTX CRO FEP RAD CXM CAZ CTX CRO FEP Ceazidime Tobramycin Cefotaxime Fosfomycin MOX LVX Ceriaxone Cefepime Cephalosporin Cephalosporin Aminoglycoside + carbapenem MOX + cephalosporin LVX + cephalosporin Amikacin Imipenem Gentamycin Meropenem Tobramycin Fosfomycin Carbapenem + cephalosporin Imipenem Cephradine Meropenem Cefuroxime Ceazidime Cefotaxime Ceriaxone Cefepime Fluoroquinolone + carbapenem Ofloxacin Imipenem Levofloxacin Meropenem Sparfloxacin Moxifloxacin (a) (b) (c) 600 600 0.132 0.265 0.126 0.126 0.126 0.126 500 500 400 400 300 300 200 200 MIC μ g/ml ± SD MIC 100 μ g/ml ± SD MIC 100 0 0 OFX LVX MOX SPX OFX LVX MOX SPX MEM IPM Fluoroquinolones Fluoroquinolones MEM + fluoroquinolones IPM + fluoroquinolones (d) (e)

Figure 3: Time-Kill kinetics on antibiotics individually and collaterally. (a) Levofloxacin-ceftazidime combination with 8-fold growth reduction compared to most active monotherapy. (b) <2-log 10 difference in killing with amoxicillin-clavulanic acid-cephradine com- bination therapy compared to most active single treatment shows lack of synergy. (c) Amoxicillin-clavulanic acid-ofloxacin combination is collaterally indifferent as decrease in bacterial growth is <2-log 10. (d) Amoxicillin-clavulanic acid-tobramycin combination shows synergy because of >2-log 10 growth reduction. (e) Collateral sensitivity is shown in piperacillin-tazobactam-tobramycin combination.

1.20E + 01 1.20E + 01 1.00E + 01 1.00E + 01 8.00E + 00 8.00E + 00 6.00E + 00 6.00E + 00 4.00E + 00 4.00E + 00 2.00E + 00 2.00E + 00 Log CFU/mL Log CFU/mL 0.00E + 00 0.00E + 00 –2.00E + 00 –2.00E + 00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time (hours) Time (hours)

Levofloxacin Amoxicillin/clavulanic acid Ceazidime Cephradine Levofloxacin-ceazidime Amoxicillin/clavulanic acid-cephradine (a) (b) Figure 4: Continued. 8 International Journal of Microbiology

1.20E + 01 1.20E + 01 1.00E + 01 1.00E + 01 8.00E + 00 8.00E + 00 6.00E + 00 6.00E + 00 4.00E + 00 4.00E + 00 2.00E + 00 2.00E + 00 Log CFU/mL 0.00E + 00 Log CFU/mL 0.00E + 00 –2.00E + 00 –2.00E + 00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time (hours) Time (hours)

Ofloxacin Amoxicillin/clavulanic acid Amoxicillin/clavulanic acid Tobramycin Amoxicillin/clavulanic acid-ofloxacin Amoxicillin/clavulanic acid-tobramycin (c) (d) 1.20E + 01 1.00E + 01 8.00E + 00 6.00E + 00 4.00E + 00 2.00E + 00

Log CFU/mL 0.00E + 00 –2.00E + 00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time (hours)

Tobramycin Piperacillin/tazobactam Piperacillin/tazobactam-tobramycin (e)

Figure 4: Collateral relationship network of antibiotic combinations from diverse subclasses on MRSA LR-2. produce better results than the ones we screened. However, Abbreviations the main objective of our study to test and identify com- binations from diverse subclasses that interacted synergis- MRSA: Methicillin-resistant Staphylococcus aureus tically, belonged to lower generations, produced bactericidal XDR: Extremely drug resistant effects, and overcome the resistance for first-line antibiotics VRE: Vancomycin-resistant Enterococcus species that could not be used anymore was achieved. FDA: Food and Drug Administration Although coadministration of drugs is a promising MIC: Minimum inhibitory concentration approach to mitigate rapid evolution of antibiotic resistance, FICI: Fractional inhibitory concentration index it may also lead to production of unexpected and unwanted MHB: Muller-Hinton Broth outcomes as reported previously [39]. Although the FIC CLSI: Clinical and Laboratory Standards Institute index was determined to ensure that a synergistic rela- RAD: Cephradine tionship exists between selected antibiotics when used in CXM: Cefuroxime combination, further investigations may help optimize the CAZ: Ceftazidime management of antibiotic combinations. CTX: Cefotaxime CRO: Ceftriaxone FEP: Cefepime 5. Conclusion AK: Amikacin We report that pairwise LVX/CAZ combination allows us to CN: Gentamycin use first-line antibiotics that not only produce bactericidal TOB: Tobramycin effects against XDR MRSA but also address need of time by FOS: Fosfomycin extending the lifespan of existing antibiotics. )is combi- OFX: Ofloxacin nation will be immediately available for use since these drugs LVX: Levofloxacin have already been approved by the FDA. Resistance to SPX: Sparfloxacin antibiotic combination though inevitable, evidence-based MOX: Moxifloxacin synergistic studies might diminish the emergence of resis- MEM: Meropenem tance. For future prospects, we propose that these combi- IPM: Imipenem nations should be further tested in vivo to determine if these AUG: Amoxicillin/clavulanic acid amalgams are as effective in vivo as they were in vitro. We TZP: Piperacillin/tazobactam. also suggest that investigations should be carried out on how these combinations, where target proteins of the antibiotics Data Availability involved in amalgam, work synergistically at molecular and systemic level to give a better insight. )is will help predict Data produced and analyzed during this study are included better and robust combinations in future. in this article. International Journal of Microbiology 9

Conflicts of Interest [11] L. K. Yaw, J. O. Robinson, and K. M. Ho, “A comparison of long-term outcomes after meticillin-resistant and meticillin- )e authors declare no conflicts of interest. sensitive Staphylococcus aureus bacteraemia: an observational cohort study,” /e Lancet Infectious Diseases, vol. 14, no. 10, pp. 967–975, 2014. Authors’ Contributions [12] Y. Bhusal, C. M. Shiohira, and N. Yamane, “Determination of in vitro synergy when three antimicrobial agents are combined SN conducted the research and prepared the manuscript. against tuberculosis,” International Journal of MA collected the samples. SV and US carried out the study. Antimicrobial Agents, vol. 26, no. 4, pp. 292–297, 2005. DG revised and reviewed the study. SA and KM designed, [13] Y. Cai, D. Chai, R. Wang, B. Liang, and N. Bai, “ reviewed, and approved the research. resistance of : clinical reports, mechanisms and antimicrobial strategies,” Journal of Anti- microbial , vol. 67, no. 7, pp. 1607–1615, 2012. 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